Pharmaceutical composition comprising squalene epoxidase inhibitor and macrolide immunomodulator

ABSTRACT

Synergistic combinations of a squalene epoxidase inhibitor such as terbinafine and a macrolide T-cell immunomodulator or immunosuppressant such as 33-epichloro, 33-desoxyascomycin are provided, which are useful in particular in the treatment of diseases involving fungal or suspected fungal infection, for immunomodulation or immunosuppression in conditions in which fungal or suspected fungal colonisation of e.g. the skin plays a role, such as atopic dermatitis and seborrhoeic dermatitis, and in situations of fungal resistance.

The invention relates to pharmaceutical compositions, for use inparticular against fungal infections.

It concerns a pharmaceutical composition comprising a squalene epoxidaseinhibitor in combination with a macrolide T-cell immunomodulator orimmunosuppressant.

While an antifungal activity is known for various macrolide T-cellimmunomodulators and immunosuppressants, and some such macrolides haveeven first been disclosed in the literature as antifungals, e.g.ascomycin and rapamycin, their antifungal activity has been viewedmostly as being largely coincidental with their immunomodulatingproperties, and further, no common pattern was known, the structural andmechanistic similarities between these compounds having been discoveredonly long after their original isolation. In particular, notwithstandingthe antifungal heritage of macrolide T-cell immunomodulators andimmunosuppressants, no significant reports have appeared in theliterature on any potentiating effect on the antifungal activity ofsqualene epoxidase inhibitors, nor are synergistic pharmaceuticalcompositions comprising these two classes of drugs in combination known.

It has now been found that, surprisingly, when used in combination,squalene epoxidase inhibitors and macrolide T-cell immunomodulators andimmunosuppressants act synergistically, resulting in a potentiation ofpharmacological activity, so that effective beneficial, especiallyantifungal activity is seen upon co-administration at dosages whichwould be well below the effective dosages individually. Further, withantifungals other than squalene epoxidase inhibitors, in particularazole 14α-methyldemethylase inhibitors, no significant positiveinteraction with such macrolides is seen, or interaction to a muchsmaller degree, or even antagonism, as with e.g. fluconazole.

The invention thus concerns novel pharmaceutical compositions comprisinga squalene epoxidase inhibitor in association or combination with amacrolide T-cell immunomodulator or immunosuppressant, hereinafterbriefly named “the compositions of the invention”.

A macrolide T-cell immunomodulator or immunosuppressant is to beunderstood herein as being a T-cell immunomodulator or T-cellimmunosuppressant which has a macrocyclic compound structure including alactone or lactam moiety. While it preferably has at least some T-cellimmunomodulating or immunosuppressant activity, it may also exhibitconcomitantly or predominantly further pharmaceutical properties, suchas anti-inflammatory activity.

The compositions of the invention may be adapted for systemic, e.g. oralor intravenous, or topical use; preferably topical use. They are usefulfor the known indications of the particular active agents incorporatedtherein. They are particularly indicated for use in diseases involvingfungal or suspected fungal infections, e.g. by yeasts such as Candida orMalassezia (Pityrosporum) spp., or dermatophyte filamentous fungi suchas Microsporum spp; or for use in conditions in which fungal orsuspected fungal colonisation of e.g. the skin plays a role, optionallyin connection with an inflammatory component or inflammatorycomplications, such as atopic dermatitis and seborrhoeic dermatitis, orin situations of fungal resistance.

A suitable squalene epoxidase inhibitor is for example a thiocarbamateantifungal such as tolnaftate or tolciclate, or an aryl- orheteroarylmethylamine antifungal, preferably of the allyl- orbenzylamine class of antifungals, e.g. as described in GB 1, 579, 879,EP 896, EP 24587, GB 2, 116, 171, GB 2, 185, 980, EP 164697, EP 221781and EP 421302. It is in particular naftifine (Exoderil®) or butenafine(Mentax®), preferably terbinafine (Lamisil®), i.e.(E)-N-methyl-N-(1-naphthylmethyl)-6,6-dimethylhept-2-en-4-amin offormula I

in free form or salt form, particularly hydrochloride acid addition saltform, disclosed as Example 16 in EP 24587.

A suitable macrolide T-cell immunomodulator or immunosuppressant is forexample an FKBP12-binding calcineurin inhibitor or mitogen-activatedkinase modulator or inhibitor, in particular an asco- or rapamycin. Itpreferably is an ascomycin. While the macrolide preferably has at leastsome calcineurin- or mitogen-activated kinase modulating or inhibitingactivity, it may also exhibit concomitantly or predominantly furtherpharmaceutical properties, such as antiinflammatory activity. Itpreferably is a compound, e.g. an ascomycin, having rather long-actingactivity relatively to other members of the same structural class, e.g.it is degraded metabolically slowly to inactive products.

An asco- or rapamycin is to be understood as asco- or rapamycin as such,or a derivative thereof. A derivative is to be understood as being anantagonist, agonist or analogue of the parent compound which retains thebasic structure and modulates at least one of the biological, forexample immunological properties of the parent compound.

Suitable ascomycins are e.g. as described in EP 184162, EP 315978, EP323042, EP 423714, EP 427680, EP 465426, EP 474126, WO 91/13889, WO91/19495, EP 484936, EP 523088, EP 532089, EP 569337, EP 626385, WO93/5059 and WO 97/8182;

in particular:

-   -   ascomycin;    -   tacrolimus (FK506; Prograf®);    -   imidazolylmethoxyascomycin (WO 97/8182 in Example 1 and as        compound of formula I);    -   32-O-(1-hydroxyethylindol-5-yl)ascomycin (L-732531)        (Transplantation 65 [1998] 10-18, 18-26, on page 11, FIG. 1; and    -   (32-desoxy,32-epi-N1-tetrazolyl)ascomycin (ABT-281) (J. Invest.        Dermatol. 12 [1999] 729-738, on page 730, FIG. 1);        preferably:    -   {1R,5Z,9S,12S-[1E-(1R,3R,4R)],13R,14S,17R,18E,21S,23S,24R,25S,27R}-17-ethyl-1,14-dihydroxy-12-[2-(4-hydroxy-3-methoxycyclohexyl)-1-methylvinyl]-23,25-dimethoxy-13,19,21,27-tetramethyl-11,28-dioxa-4-azatricyclo[22.3.1.0(4,9)]octacos-5,18-diene-2,3,10,16-tetraone        (Example 8 in EP 626385),    -    hereinafter referred to as “5,6-dehydroascomycin”;    -   {1E-(1R,3R,4R)]1R,4S,5R,6S,9R,10E,13S,15S,16R,17S,19S,20S}-9-ethyl-6,16,20-thihydroxy-4-[2-(4-hydroxy-3-methoxycyclohexyl)-1-methylvinyl]-15,17-dimethoxy-5,11,13,19-tetramethyl-3-oxa-22-azatricyclo[18.6.1.0(1,22)]heptacos-10-ene-2,8,21,27-tetraone        (Examples 6d and 71 in EP 569337),    -    hereinafter referred to as “ASD 732”; and    -   pimecrolimus (INN recommended) (ASM981; Elidel™), i.e        {[1E-(1R,3R,4S)]1R,9S,12S, 13R,14S,17R,18E,        21S,23S,24R,25S,27R}-12-[2-(4-chloro-3-methoxycyclohexyl)-1-methylvinyl]-17-ethyl-1,14-dihydroxy-23,25-dimethoxy-13,19,21,27-tetramethyl-11,28,dioxa-4-azatricyclo        [22.3.1.0(4,9)]octacos-18-ene-2,3,10,16-tetraone, of formula I    -    (Example 66a in EP 427680),    -    hereinafter referred to as “33-epichloro,33-desoxyascomycin”.

Suitable rapamycins are e.g. as described in U.S. Pat. No. 3,929,992, WO94/9010 and U.S. Pat. No. 5,258,389, preferably sirolimus (rapamycin;Rapamune®) and everolimus (RAD001; Certican®).

Particularly preferred are compositions of the invention comprising anarylmethylamine antifungal in combination with an ascomycin, especiallyterbinafine in combination with 33-epichloro,33-desoxyascomycin.

Preferred for use in the treatment of conditions where inflammation isinvolved are compositions of the invention wherein one or bothcomponents possess some degree of inherent anti-inflammatory activity,such as naftifine or terbinafine in combination with33-epichloro,33-desoxyascomycin.

“Treatment” as used herein includes prevention, namely prophylactic aswell as curative treatment.

Synergy is e.g. calculated as described in Example 1 hereunder or asdescribed in Berenbaum, Clin. Exp. Immunol. 28 (1977) 1, using aninteraction term to correct for differences in mechanism between the twodrugs, as described in Chou et al., Transpl. Proc. 26 (1994) 3043. Theindex of synergy is calculated as:$\frac{{dose}\quad{of}\quad A}{A_{E}} + \frac{{dose}\quad{of}\quad B}{B_{E}} + \frac{\left( {{dose}\quad{of}\quad A} \right) \times \left( {{dose}\quad{of}\quad B} \right)}{A_{E} \times B_{E}}$in which the doses of the compounds A and B represent those used in aparticular combination, and A_(E) and B_(E) are the individual doses ofA and B respectively giving the same effect. If the result is less than1, there is synergy; if the result is 1, the effect is additive; if theresult is greater than 1, A and B are antagonistic. By plotting anisobologram of dose of A/A_(E) vs. dose of B/B_(E), the combination ofmaximum synergy can be determined. The synergistic ratio expressed interms of the ratio by weight of the two compositions at synergisticamounts along the isobologram, especially at or near the point ofmaximum synergy, can then be used to determine formulations containingan optimally synergistic ratio of the two compounds.

The invention also provides products and methods for co-administrationof a squalene epoxidase inhibitor, e.g. terbinafine and a macrolideT-cell immunomodulator or immunosuppressant, e.g.33-epichloro,33-desoxyascomycin or 5,6-dehydroascomycin, atsynergistically effective dosages, e.g.:

-   -   a method of treatment or prevention of diseases involving a        fungal or suspected fungal infection, or a method for        immunomodulation or immunosuppression in a condition in which        fungal or suspected fungal colonization plays a role or in        situations of fungal resistance; in a subject suffering from or        at risk for such infection or condition, comprising        co-administering synergistically effective amounts of a        composition of the invention;    -   the use of a squalene epoxidase inhibitor in the manufacture of        a medicament for co-administration in synergistically effective        amounts with a macrolide T-cell immunomodulator or        immunosuppressant;    -   the use of a macrolide T-cell immunomodulator or        immunosuppressant in the manufacture of a medicament for        co-administration in synergistically effective amounts with a        squalene epoxidase inhibitor;    -   a kit of parts comprising a squalene epoxidase inhibitor and a        macrolide T-cell immunomodulator or immunosuppressant in        separate unit dosage forms, preferably wherein the unit dosage        forms are suitable for administration of the component compounds        in synergistically effective amounts, together with instruction        for use, optionally with further means for facilitating        compliance with the administration of the component compounds,        e.g. a label or drawings;    -   the use of a squalene epoxidase inhibitor in the manufacture of        a pharmaceutical kit which is to be used for facilitating        co-administration with a macrolide T-cell immunomodulator or        immunosuppressant;    -   the use of a macrolide T-cell immunomodulator or        immunosuppressant in the manufacture of a pharmaceutical kit        which is to be used for facilitating co-administration with a        squalene epoxidase inhibitor;    -   a squalene epoxidase inhibitor and a macrolide T-cell        immunomodulator or immunosuppressant as a combined        pharmaceutical preparation for simultaneous, separate or        sequential use, preferably in synergistically effective amounts,        e.g. for the treatment or prevention of a fungal infection, or        for immunomodulation or immunosuppression in a condition in        which fungal or suspected fungal colonization plays a role;    -   a pharmaceutical composition comprising a squalene epoxidase        inhibitor in combination or association with a macrolide T-cell        immunomodulator or immunosuppressant, e.g. in synergistically        effective amounts, together with at least one a pharmaceutically        acceptable diluent or carrier, e.g. for use in treatment or        prevention of a fungal infection, or for immunomodulation or        immunosuppression in a condition in which fungal or suspected        fungal colonization plays a role, or in a situation of fungal        resistance; and    -   a process for the preparation of a composition of the invention        comprising mixing a squalene epoxidase inhibitor and a macrolide        T-cell immunomodulator or immunosuppressant, in combination or        association with at least one pharmaceutically acceptable        diluent or carrier.

By “synergistically effective amounts” is meant an amount of squaleneepoxidase inhibitor and an amount of macrolide T-cell immunomodulator orimmunosuppressant which are individually below their respectiveeffective dosages for a relevant indication, but which arepharmaceutically active on co-administration, e.g. in a synergisticratio, for example as calculated above. Furthermore, “synergisticallyeffective amounts” may mean an amount of squalene epoxidase inhibitorand an amount of macrolide T-cell immunomodulator or immunosuppressantwhich are individually equal to their respective effective dosages for arelevant indication, and which result in a more than additive effect.

The molar amount of squalene epoxidase inhibitor present is from roughlysimilar to, to significantly more than the amount of macrolide T-cellimmunomodulator or immunosuppressant, preferably twice as much or more.Synergistic ratios of squalene epoxidase inhibitor to macrolide T-cellimmunomodulator or immunosuppressant by weight are thus suitably fromabout 1:10 to about 50:1, preferably from about 1:5 to about 20:1, mostpreferably from about 1:1 to about 15:1, e.g. about 12:1.

The compositions of the invention can be administered as a freecombination, or the drugs can be formulated into a fixed combination,which greatly enhances the convenience for the patient.

Absolute dosages of the compounds will vary depending on a number offactors, e.g. the individual, the route of administration, the desiredduration, the rate of release of the active agent and the nature andseverity of the condition to be treated. For example, the amount ofactive agents required and the release rate thereof may be determined onthe basis of known in vitro and in vivo techniques, determining how longa particular active agent concentration in the blood plasma remains atan acceptable level for a therapeutic effect.

For example, in prevention and treatment of fungal or suspected fungalinfection, an initial dosage of about 2-3 times the maintenance dosageis suitably administered, followed by a daily dosage of about 2-3 timesthe maintenance dosage for a period of from one to two weeks, andsubsequently the dose is gradually tapered down at a rate of about 5%per week to reach the maintenance dosage. In general, synergisticallyeffective amounts of terbinafine and 33-epichloro,33-desoxyascomycin onoral administration for use in prevention and treatment of fungaldiseases in larger animals, e.g. man, are amounts of terbinafine of upto about 50 mg/kg/day, e.g. from about 0.25 mg/kg/day to about 50mg/kg/day, preferably about 2.5 mg/kg/day, in combination orco-administration with amounts of 33-epichloro,33-desoxyascomycin of upto about 2 mg/kg/day, e.g. from about 0.01 mg/kg/day to about 2mg/kg/day, preferably about 0.5 mg/kg/day, in a synergistic ratio, asdescribed. Suitable unit dosage forms for oral co-administration ofthese compounds thus may contain on the order of from about 10 mg toabout 3000 mg, preferably about 50 mg to about 500 mg of terbinafine,and from about 0.5 mg to about 100 mg, preferably about 3 mg to about 30mg of 33-epichloro,33-desoxyascomycin. The daily dosage for oraladministration is preferably taken in a single dose, but may be spreadout over two, three or four dosages per day. For i.v. administration,the effective dosage is lower than that required for oraladministration, e.g. about one fifth the oral dosage.

By “co-administration” is meant administration of the components of thecompositions of the invention together or at substantially the sametime, e.g. within fifteen minutes or less, either in the same vehicle orin separate vehicles, so that upon oral administration, for example,both compounds are present simultaneously in the gastrointestinal tract.Preferably, the compounds are administered as a fixed combination.

The compositions of the invention include compositions suitable foradministration by any conventional route, in particular compositionssuitable for administration either enterally,. for example, orally, e.g.in the form of solutions for drinking, tablets or capsules, orparenterally, e.g. in the form of injectable solutions or suspensions;or topically, e.g. for the treatment of fungal conditions of the skin ormucosae, e.g. in the form of a dermal cream, ointment, ear drops,mousse, shampoo, solution, lotion, gel, emulgel or like preparation,e.g. in a concentration of from about 0.1% to about 2% by weight of eachcomponent, especially in combination or association with penetrationenhancing agents, as well as for application to the eye, e.g. in theform of an ocular cream, gel or eye-drop preparation, for treatment offungal or suspected fungal conditions of the lungs and airways, e.g. inthe form of inhalable compositions, and for mucosal application, e.g. inthe form of vaginal tablets.

The compositions of the invention are suitably emulsions,microemulsions, emulsion preconcentrates or microemulsionpreconcentrates, or solid dispersions, especially water-in-oilmicroemulsion preconcentrates or oil-in-water microemulsions, comprisingthe squalene epoxidase inhibitor and the macrolide T-cellimmunomodulator or immunosuppressant in a synergistic ratio.

The compositions of the invention can be prepared in conventionalmanner, e.g. by mixing a squalene epoxidase inhibitor and a macrolideT-cell immunomodulator or immunosuppressant, in combination orassociation with at least one pharmaceutically acceptable diluent orcarrier.

The active agent components may be in free form or pharmaceuticallyacceptable salt form as appropriate.

The following Examples illustrate the invention. All temperatures are indegrees Celsius. The compounds are in free, i.e. neutral or base formunless specified otherwise. The following abbreviations are used:

BSA = bovine serum albumin cfu = colony-forming units FKBP12 =FK-binding protein 12 MIC = minimum inhibitory concentration MOPS =3-(N-morpholino)propanesulfonic acid

EXAMPLE 1 Synergism in Yeast (Candida)

Combination studies are performed with Candida species, using the NCCLSstandard assay (National Committee for Clinical Laboratory Standards[1995], Reference method for broth dilution antifungal susceptibilitytesting of yeasts (Tentative Standard M27-T, NCCL, Villanova, Pa.,U.S.A.), modified as follows: the assay is effected in RPMI 1640 mediumwithout NaHCO₃ but with L-glutamine buffered with MOPS at pH 7.0 at 37°.The inoculum is 2.5×10³ cfu/ml. Incubation times are 48 hours. The MICis defined as the lowest drug concentration causing 80% inhibition offungal growth.

The reference test compound (either terbinafine or fluconazole) istested in a full concentration range in the absence or presence of theother test drug at 1, 2, 4, and 8 μg/ml.

Interaction between the drugs is analysed by calculation of theFractional Inhibitory Concentration Index (FICI), defined as:FICI=[(MIC A in combination)/MIC A]+[MIC B in combination]/MIC B]

Interpretation of FICI in terms of interaction between the two drugs isas follows: FICI=0.5: synergy; <0.5<FICI=1: additive; 1<FICI=2:indifferent; 2<FICI: antagonism (in practical terms, synergy by thisdefinition means that the MICs of both drugs are reduced by at least 2dilution steps when used in combination).

The ability of the four test compounds 33-epichloro,33-desoxyascomycin,5,6-dehydroascomycin, ascomycin and tacrolimus to influence theantifungal activity of terbinafine and fluconazole was examined in aCandida albicans isolate susceptible to both drugs, and in one isolateof Candida krusei which is resistant to terbinafine and poorlyresponsive to fluconazole. The test compounds alone are completelyinactive against both strains. The Table summarises the patterns ofinteractions which have been obtained:

TABLE 1 Interaction of macrolide 33-epichloro,33-desoxyascomycin withterbinafine or with fluconazole in Candida yeast Interaction Interactionwith terbinafine^(a) with fluconazole^(a) Compound C. albicans C. kruseiC. albicans C. krusei 33-Epichloro,33- + − − − desoxy-ascomycin5,6-Dehydro- + − − − ascomycin Ascomycin + (* − − Tacrolimus + (* − −^(a)The symbols refer to the interpretation of the FICI value, asdescribed in this Example, and mean: (*: synergy; +: additive; −:indifferent/antagonistic)

With terbinafine as reference compound, all four test compounds showpotent interaction against Candida albicans, while ascomycin andtacrolimus are powerfully synergistic with terbinafine against Candidakrusei, and 33-epichloro,33-desoxy-ascomycin and 5,6-dehydroascomycinare also strongly positive. A different pattern is seen withfluconazole, which shows no potent synergy, and is antagonistic in manycases.

EXAMPLE 1a Synergism in Dermatophyte (Microsporum canis)

Minimum Inhibitory Concentrations (MIC) against dermatophytes aredetermined in 96-well assay trays by a modification of the NCCLSmicrodilution procedure M38-P (National Committee for ClinicalLaboratory Standards, Reference method for broth antifungalsusceptibility testing of conidium-forming filamentous fungi; NCCLSdocument M38-P; NCCLS, Wayne, Pa., USA [1998]), as detailed in H. A.Norris et al., J. Am. Acad. Dermatol. 40 [1999] S9-S13). The MIC isdefined as the lowest drug concentration causing 80% inhibition offungal growth.

Combination studies are performed in a checkerboard design using theabove assay to provide a matrix of all possible dose combination of thetwo drugs within the required concentration range. The matrix structureis provided by the microtiter trays used for the assay. The terbinafinedilution series is arranged on the y-axis (in columns) and the partnerdrug on the x-axis (in rows). In each row, well no. 1 contains thesterility control, well no. 2 contains terbinafine hydrochloride alone,wells no. 3 to no. 11 contain the dilution series of the partner drug33-epichloro,33-desoxyascomycin, and well no. 12 contains the growthcontrol. In each column, well A contains the partner drug alone andwells B to H contain the dilution series of terbinafine hydrochloride.

The nature of the interaction between the two drugs is definedquantitatively by means of the Fractional Inhibitory Concentration Index(FICI), which is calculated by the following formula:FICI=[(MIC A in combination)/MIC A]+[(MIC B in combination)/MIC B].

Interpretation of the FICI is:

-   ≦0.5: synergy;-   >0.5 but ≦1: additive;-   >1 but ≦2: indifferent;-   >2: antagonism.

In practice, synergy calculated in this way is equivalent to a reductionof at least 2 dilution steps in the MIC of each drug when they arecombined.

The results obtained with Microsporum canis (strain NFI 5167) areindicated in Table 2:

TABLE 2 Interaction of macrolide 33-epichloro,33-desoxyascomycin withterbinafine hydrochloride in dermatophyte Microsporum canis MIC singledrug MIC combination terbinafine partner terbinafine partner FICIInterpretation 0.031 64 0.001 16.000 0.28 synergy

EXAMPLE 2 Tablet

A tablet for oral use with granulated terbinafine hydrochloride and33-epichloro,33-desoxyascomycin in form of a solid dispersion isprepared in conventional manner, in a 600 mg dosage, and contains thefollowing ingredients:

Component Amount (mg) 33-Epichloro,33-desoxyascomycin 20.0 Terbinafinehydrochloride 281.25 (corresponds to 250 mg free base) Silicium dioxidecolloidal 1.95 (Aerosil 200) Microcrystalline cellulose 48.30 Sodiumcarboxymethyl starch 35.10 Hydroxypropylmethyl cellulose 3 cps 81.70Poloxamer 188 10.00 Lactose, anhydrous 67.50 Crospovidone 50.00Magnesium stearate 4.20 Total 600.00

EXAMPLE 3 Cream

A cream with dissolved terbinafine base is prepared in conventionalmanner with 33-epichloro,33-desoxyascomycin, both in a 1% w/wconcentration, and contains the following ingredients:

Component Amount (g) 33-Epichloro,33-desoxyascomycin 1.00 Terbinafinebase 1.00 Triglycerides, medium chain 15.00 Oleyl alcohol 10.00 Sodiumcetylstearyl sulfate 1.00 Cetyl alcohol 4.00 Stearyl alcohol 4.00Glyceryl monostearate 2.00 Benzyl alcohol 1.00 Propylene glycol 5.00Citric acid 0.05 Sodium hydroxide 0.02 Water 55.93 Total 100.00

EXAMPLE 4 Ointment

An ointment with terbinafine hydrochloride and33-epichloro,33-desoxyascomycin in suspended form is prepared inconventional manner in a 1% w/w concentration, and contains thefollowing ingredients:

Component Amount (g) 33-Epichloro,33-desoxyascomycin 1.00 Terbinafinehydrochloride 1.125 Mineral oil 45.00 Petrolatum 42.875 Microcrystallinewax 10.00 Total 100.00

EXAMPLE 5 Vaginal Tablet

A tablet for vaginal use with granulated terbinafine hydrochloride and33-epichloro,33-desoxyascomycin is prepared in conventional manner, in a1600 mg dosage, and contains the following ingredients:

Component Amount (mg) 33-Epichloro,33-desoxyascomycin 20.0 Terbinafinehydrochloride 281.25 (corresponds to 250 mg free base) Lactosemonohydrate 1004.75 Sodium carboxymethyl starch 96.00Hydroxypropylmethyl cellulose 3 cps 54.00 Corn starch 112.0 Magnesiumstearate 32.00 Total 1600.00

Terbinafine in Examples 2 to 5 may be replaced by a molar equivalentamount of tolnaftate, tolciclate, naftifine or butenafine.

33-Epichloro,33-desoxyascomycin in Examples 2 to 5 may be replaced by amolar equivalent amount of ascomycin, tacrolimus,imidazolylmethoxyascomycin, 32-O-(1-hydroxyethylindol-5-yl)ascomycin,(32-desoxy,32-epi-N1-tetrazolyl)ascomycin, 5,6-dehydroascomycin, ASD732, sirolimus or everolimus.

1. A pharmaceutical composition comprising a combination of a squaleneepoxidase inhibitor selected from a thiocarbamate antifungal or anarylmethylamine or heteroarylmethylamine antifungal and a macrolideT-cell immunomodulator or immunosuppressant which has a macrocycliccompound structure including a lactone or lactame moiety and is anascomycin or rapamycin, as active ingredients, together with at leastone pharmaceutically acceptable diluent or carrier, wherein saidsqualene epoxidase inhibitor and said macrolide T-cell immunomodulatoror immunosuppressant of said composition can be administered atsubstantially the same time or in combination.
 2. A compositionaccording to claim 1 comprising terbinafine and 33-epichloro,33-desoxyascomycin.
 3. A method of treatment of a disease involvingfungal infection, or a method for immunomodulation or immunosuppressionin a condition in which fungal colonization plays a role or in asituation of fungal resistance, in a subject suffering from or at riskfor such infection or condition, comprising administering asynergistically effective amount of a composition according to claim 1to a subject in need of such treatment.
 4. A process for the preparationof a composition according to claim 1 comprising mixing the squaleneepoxidase inhibitor and the macrolide T-cell immunomodulator orimmunosuppressant with at least one pharmaceutically acceptable diluentor carrier.
 5. A kit of parts comprising a squalene epoxidase inhibitorselected from thiocarbamate antifungal or an aryl- orheteroarylmethylamine antifungal and a macrolide T-cell immunomodulatoror immunosuppressant which has a macro cyclic compound structureincluding a lactone or lactame moiety and is an asco- or rapamycin, inseparate unit dosage forms.
 6. A composition according to claim 1wherein said squalene epoxidase inhibitor is terbinafine.
 7. Acomposition according to claim 1 wherein said macrolide T-cellimmunomodulator or immunosuppressant is 33-epichloro,33-desoxyascomycin.